CS271293B1 - Mouse lymphocyte hybridoma vu t51/3 - Google Patents
Mouse lymphocyte hybridoma vu t51/3 Download PDFInfo
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- CS271293B1 CS271293B1 CS888157A CS815788A CS271293B1 CS 271293 B1 CS271293 B1 CS 271293B1 CS 888157 A CS888157 A CS 888157A CS 815788 A CS815788 A CS 815788A CS 271293 B1 CS271293 B1 CS 271293B1
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- Prior art keywords
- hybridoma
- type
- herpes simplex
- monoclonal antibody
- virus
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- 210000004408 hybridoma Anatomy 0.000 title claims abstract description 14
- 210000004698 lymphocyte Anatomy 0.000 title claims description 3
- 241001529936 Murinae Species 0.000 claims abstract description 3
- 241000700588 Human alphaherpesvirus 1 Species 0.000 claims description 9
- 241000700605 Viruses Species 0.000 abstract description 5
- 238000003127 radioimmunoassay Methods 0.000 abstract description 5
- 239000000427 antigen Substances 0.000 abstract description 4
- 102000036639 antigens Human genes 0.000 abstract description 4
- 108091007433 antigens Proteins 0.000 abstract description 4
- 238000002965 ELISA Methods 0.000 abstract description 2
- 239000000463 material Substances 0.000 abstract description 2
- 238000004458 analytical method Methods 0.000 abstract 1
- 230000000527 lymphocytic effect Effects 0.000 abstract 1
- 230000003612 virological effect Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 12
- 241000699666 Mus <mouse, genus> Species 0.000 description 5
- 241000700584 Simplexvirus Species 0.000 description 5
- 206010003445 Ascites Diseases 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 206010035226 Plasma cell myeloma Diseases 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 201000000050 myeloid neoplasm Diseases 0.000 description 3
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000701074 Human alphaherpesvirus 2 Species 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 210000003200 peritoneal cavity Anatomy 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 239000006145 Eagle's minimal essential medium Substances 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 239000005662 Paraffin oil Substances 0.000 description 1
- 230000003322 aneuploid effect Effects 0.000 description 1
- 208000036878 aneuploidy Diseases 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 239000012568 clinical material Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 210000004754 hybrid cell Anatomy 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 241001529453 unidentified herpesvirus Species 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
Vynález sa týká nového hybridómu, t»j· hybridného jednobunkového organizmu, zostrojeného fúziou myšej myelómovej buňky Sp2/O a myše) slezinovej lymfoidnej buňky, produkujúcej protilátku voči virusu Herpes simplex typ 1.The invention relates to a novel hybridoma, i.e. a hybrid one-cell organism constructed by fusing a mouse myeloma cell Sp2 / O and a mouse spleen lymphoid cell producing an antibody to Herpes simplex virus type 1.
U virusu Herpes simplex rozoznávame dva antigénne odlišné typy Herpes simplex typ 1 a typ 2« Doteraz sa protilátky (antiséra) voči virusu Herpes simplex typ 1 připravovali imunizáciou pokusných zvierat, najčastejšie králikov, purifikovaným alebo nepurifikovaným vírusom resp· niektorým izolovaným proteínom /Forghani B., Schmidt N., Lennette E. H·: Solid phase rádioimmunoassay for identitication of herpesvirus hominis types 1 and 2 from clinical materiál· Appl. Microbiol· 28, /1974/ 661 - 667* Dreesman G. R., Watson 0. 0., Courtney R. □·, Adam E·, Melnick □· L.: Oetection of herpesvirus type- 4 In Herpes simplex virus, we recognize two antigenically different types of Herpes simplex type 1 and type 2. , Schmidt N., Lennette E. H: Solid phase radioimmunoassay for identification of herpesvirus hominis types 1 and 2 from clinical material Appl. Microbiol · 28, / 1974/661 - 667 * Dreesman GR, Watson 0. 0, Courtney R. Adam, Adam E Mel, Melnick L L .: Oetection of herpesvirus type- 4
-specific antibody by microsolid-phase radioimmunometric assay* Intervirology 12, /1979/ 115 - 119)· Sérum takto imunizovaných zvierat, odobrané po viacerých dávkách antigénu, slúžilo ako zdroj protilátok tzv· typovošpecifických, ktoré sa využívali na dokaž antigénu virusu Herpes simplex typ 1 v základnom výskume a v imunodiagnostickej praxi· Vzhladom na to, že virusy Herpes simplex typ 1 a 2 obsahujú nielen typovošpecifické, ale aj typovo spoločné antigénne determinanty, takto připravené antiséra voči virusu Herpes simplex obsahujú vysoké hladiny typovospoločných protilátok, ktoré slažujú resp· úplné znemožňujú správné určenie typu infikujúceho virusu pri diagnostickom testovaní· TypovospoloČné protilátky sa obvykle odstranujú zo sér vysycovánim s vírusom Herpes simplex typ 2, čo je procedúra velmi náročná na materiál a naviac len v malom počte prípadov úspěšná· Výrobné šarže konvenčných typovošpecifických antisér sa dajú lažko Standardizoval a bývajú v Sirokom rozmedzi kvality· V poslednom čase sa s úspěch om používájú na typizáciu virusu Herpes simplex typ 1 monoklonálne protilátky tzv· typovošpecifické, ktoré sú schopné detegoval infekciu vírusom Herpes simplex typ 1 v кliniekom materiále·The serum of such immunized animals, taken after multiple doses of antigen, served as a source of so-called type-specific antibodies which were used to prove the Herpes simplex virus type antigen. · Since Herpes simplex viruses type 1 and 2 contain not only type-specific, but also type-common antigenic determinants, the herpes simplex virus antisera thus prepared contain high levels of type-common antibodies that reduce or prevent completely Correctly identifying the type of infecting virus in diagnostic testing · Type-common antibodies are usually removed from Herpes simplex type 2 virus sera, which is a very material-intensive procedure and, in a small number of cases, successful · Production batches of conventional typespecific h Antisera are easy to standardize and live in a wide quality range · Recently, monoclonal antibodies, so-called type-specific monoclonal antibodies that are capable of detecting Herpes simplex type 1 virus infection, have been successfully used to clone the virus.
Uvedené nevýhody doteraz používaných postupov sa nevyskytnú, ak je к dispozic i i hybridómova banková línia produkujúca typovošpecifickú monoklonálnu protilátku voči virusu Herpes simplex typ 1, ktorá Je uložená v zbierke hybridómov Virologického ústavu SAV, Mlýnská dolina 1, Bratislava pod označením VÚ T51/3.The above disadvantages of the methods used so far do not occur if there is also a hybridoma bank line producing a type-specific monoclonal antibody against Herpes simplex virus type 1, which is stored in the collection of hybrids of the Institute of Virology of the Slovak Academy of Sciences, Mlýnská dolina 1, Bratislava.
Uvedený hybridám bol získaný spósobom známým z odbornej literatúry (Kohler, G·, Milstein, C·; Continuous cultures of fused cells secreting antibody of predefined specifity· Nátur, 256 /1975/, 495·, Gerhard, W.: Fusion of cells in suspension and outgrowth of hybride in conditioned medium· Monoclonal antibodies: A new dimension in Biological analyses· Kennett R· H. a spol·, eds New York, plenům Press /1980/, 37C)· Hybridné buňky získané po fúzi i myších myelómových Sp2/0 buniek a buniek získaných zo sleziny myši BALB/c imunizované) extraktom buniek infikovaných vírusom Herpes simplex typ 1, bol i klonované, a po otestovaní bol vybraný klon VÚ T51/3· Výhodou hybridómu je, že produkuje homogénnu protilátku, tzv. monoklonálnu protilátku, ktorá Je schopná Specificky reagoval 8 vírusom Herpes simplex typ 1. Hybridám vil T51/3 možno kultivoval in vitro v médiach vhodných pre živočišné buňky alebo in vivo v perritoneálnej dutině myši kmeňa BALB/c· Z konzerv zmrazených buniek uchovaných v kvapalnom dusíku, možno začal produkciu protilátky bez óalŠej imunizácie zvierala antigénom· , i Příklad:The hybrids were obtained in a manner known from the literature (Kohler, G, Milstein, C; Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity, Nature, 256 (1975), 495), Gerhard, W., Fusion of Cells in · Monoclonal antibodies: A new dimension in Biological analyzes · Kennett R.H. et al., eds New York, Diapers Press (1980), 37C) · Hybrid cells obtained after fusion of murine myeloma Sp2 (0 cells and cells obtained from the spleen of BALB (c mice immunized) with an extract of cells infected with Herpes simplex virus type 1, were also cloned, and after testing a clone of TU T51 / 3 was selected. a monoclonal antibody capable of specifically reacting with Herpes simplex virus type 8 virus. T51 / 3 hybrid hybrids can be cultured in vitro in animal cell media or in vivo in the perritoneal cavity of a BALB / c mouse. From canned frozen cells stored in liquid nitrogen , the production of an antibody without further immunization with an antigen may have begun.
Za účelom získania váčšieho množstva monoklonálnej protilátky νΰ T51/3 kultiváciou hybridómov buniek in vivo, 5xl06 buniek sa aplikovalo do peritoneálnej dutiny myší. Pre lepSie uchytenie buniek bola myš 15 dní před aplikáciou buniek premedikovaná parafínovým olejom /0,5 ml antraperitoneálne na 1 myš/, po 10 dnoch rastu hybridómu v peritoneálnej dutině, bola myš zabitá a vyprodukovaná ascitická tekutina odobraná. Týmto postupom možno priemerne získat asi 7 ml ascitickej tekutiny obsahujúcej 8 mg/ml protilátky. Ascitická tekutina obsahujúca produkt hybridómu VÚ T51/3 vykazovala Specifická vazbu к virusu Herpes simplex typ 1 v rádioimunoanalytickom teste /RIA/, v imunoenzymatickej analýze /ELISA/ a v imunofluorescenčnom teste /IF/.In order to obtain a greater amount of monoclonal antibody ν51 T51 / 3 by culturing hybridomas of cells in vivo, 5x10 6 cells were injected into the peritoneal cavity of mice. For better cell attachment, mice were pre-treated with paraffin oil (0.5 ml anthraperitoneally per mouse) 15 days prior to cell application, after 10 days of hybridoma growth in the peritoneal cavity, the mouse was killed and the ascites fluid produced was collected. On average, about 7 ml of ascites fluid containing 8 mg / ml antibody is obtained by this procedure. The ascites fluid containing the hybridoma product T51 / 3 showed specific binding to Herpes simplex virus type 1 in the radioimmunoassay (RIA), immunoenzymatic assay (ELISA) and immunofluorescence assay (IF).
CS 271293 8181
Buňky hybridómu VÚ T51/3 rastů in vitro ako polosuspenzná kultúra. Majů gulatý tvar a velkost charakteristická pre myelómove buňky· Obsahujú fúzované buňkové jadrá, sú aneuploídne· Buňky hybridómu VÚ T51/3 majú ultraštruktúrny obraz typických myelomových buniek, kde prevažujúcou organelou su volné a na membránu viazané polyribozómy. Základným kultivačným médiom je Dulbeccova modifikacia Eagleovho minimálneho esenciólneho média (Dulbecco, R., Freeman, G., Virology 8/1959/, 396). Toto médium, označované ako OMEM, je pre kultiváciu hybridómu doplněné gentamycínom a inaktivovaným prekolostrálnym telecím sérom (10%, Bioveta, Ivanovice na Hané)· Hybridóm je kultivovaný pri 37 °C v atmosféře 5% CO^· 3eho generačně doba je přibližné 24 h· produkovaná protilátka je monoklonály imunoglobín podtriedy igG 2b·In vitro hybridoma cells of T51 / 3 growth as a semi-suspension culture. They have a round shape and size characteristic of myeloma cells. They contain fused cell nuclei, are aneuploid. The basic culture medium is Dulbecco's modification of Eagle's minimal essential medium (Dulbecco, R., Freeman, G., Virology 8 (1959), 396). This medium, called OMEM is the cultivation of the hybridoma supplemented with gentamicin and inactivated precolostral calf serum (10%, Bioveta Ivanovice na Hane) · The hybridoma was cultured at 37 ° C in 5% CO ^ · 3 eho of recent time is about 24 h · the antibody produced is immunoglobin monoclonals of subclass igG 2b ·
Hybridóm VÚ T51/3 može byt využívaný ako zdroj protilátky voči virusu Herpes simplex typ 1, ktorá sa da použit na kvalitatívny dokaž přítomnosti virusu Herpes simplex typ 1 vo vyšetrovanom materiále, na kvantitativné stanovenie množstva infikujúceho virusu, pri vyhodnocovaní epidemiologickéj situácie a ako zdroj protilátky jedinej podtriedy (igG 2b) pre přípravu antisér Specifických pre uvedená podtriedu.The T51 / 3 hybridoma can be used as a source of antibody to Herpes simplex virus type 1, which can be used to qualitatively prove the presence of Herpes simplex virus type 1 in the material under investigation, quantitatively determining the amount of infecting virus, assessing the epidemiological situation and a single subclass (igG 2b) for preparing antisera specific for said subclass.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS888157A CS271293B1 (en) | 1988-12-09 | 1988-12-09 | Mouse lymphocyte hybridoma vu t51/3 |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS888157A CS271293B1 (en) | 1988-12-09 | 1988-12-09 | Mouse lymphocyte hybridoma vu t51/3 |
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| Publication Number | Publication Date |
|---|---|
| CS815788A1 CS815788A1 (en) | 1989-11-14 |
| CS271293B1 true CS271293B1 (en) | 1990-09-12 |
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| Application Number | Title | Priority Date | Filing Date |
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| CS888157A CS271293B1 (en) | 1988-12-09 | 1988-12-09 | Mouse lymphocyte hybridoma vu t51/3 |
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1988
- 1988-12-09 CS CS888157A patent/CS271293B1/en unknown
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| CS815788A1 (en) | 1989-11-14 |
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